A. Cloning and Vectors
The introduction of exogeneous DNA into eucaryotic cells has become one of the most powerful tools of the molecular biologist. This process requires efficient delivery of the DNA into the nucleus of the recipient cell and subsequent identification of cells that are expressing the foreign DNA.
Engineered vectors such as plasmids or bacteriophages (phages) or other DNA sequence that is able to replicate in a host cell can be used to construct cells that act as factories to produce large amounts of specific viral proteins. Recombinant plasmids will be used herein as exemplary vectors, also called cloning vehicles. See U.S. Pat. No. 4,338,397, incorporated herein by reference.
Plasmids are extrachromosomal genetic elements found in a variety of bacterial species. They are typically double-stranded, closed, circular DNA molecules. The most widely used plasmid is pBR 322, a vector whose nucleotide sequence and endonuclease cleavage sites are well known.
Nucleic acid production using plasmid or phage vectors has become very straightforward. The plasmid or phage DNA is cleaved with a restriction endonuclease and joined in vitro to a foreign DNA of choice. The resulting recombinant plasmid or phage is then introduced into a cell such as E. coli, and the cell so produced is induced to produce many copies of the engineered vector. Once a sufficient quantity of DNA is produced by the cloning vector, the produced foreign DNA is excised and placed into a second vector to produce or transcribe the protein or polypeptide encoded by the foreign gene.
Depending on the DNA (intact gene, cDNA, or bacterial gene), it may be necessary to provide eucaryotic transcription and translation signals to direct expression in recipient cells. These signals may be provided by combining the foreign DNA in vitro with an expression vector.
Expression vectors contain sequences of DNA that are required for the transcription of cloned genes and the translation of their messenger RNA's (mRNA's) into proteins. Typically, such required sequences or control elements are: (1) a promoter that signals the starting point for transcription; (2) a terminator that signals the ending point of transcription; (3) an operator that regulates the promotor; (4) a ribosome binding site for the initial binding of the cells' protein synthesis machinery; and (5) start and stop codons that signal the beginning and ending of protein synthesis.
To be useful, an expression vector should possess several additional properties. It should be relatively small and contain a strong promoter. The expression vector should carry one or more selectable markers to allow identification of transformants. It should also contain a recognition site for one or more restriction enzymes in regions of the vector that are not essential for expression.
The construction of expression vectors is, therefore, a complicated and somewhat unpredictable venture. The only true test of the effectiveness of an expression vector is to measure the frequency with which the synthesis of the appropriate mRNA is initiated. However, quantitation of mRNA is tedious, and it is often difficult to obtain accurate measurements. Other more practicable means have, therefore, been developed to detect transformation.
One such means has been to monitor synthesis of foreign proteins in transformed cells with enzymatic assays. Several marker genes have been developed for indicating that transformation has occurred.
Another means used to monitor transformation involves the use of immunological reagents. If the level of expressed protein is sufficiently high, then cytoplasmic or surface immunofluorescence with an antibody conjugated to a fluorescent dye such as fluorescein or rhodamine may be used to detect vector-specific protein expression products.
More commonly, transformed cells are cultured in the presence of radioactivity after immunoprecipitation. This approach has used Staphyloccocus aureus protein A selection of immune complexes [Kessler, (1975), J. Immunol., 115:1617-1624] and the Western blotting procedure [Renart et al., (1979), Proc. Natl. Acad. Sci. U.S.A., 76:3116-3120] to detect transformation-specific markers.
Analysis of gene expression using Simian Virus 40 (SV40) vectors is by far the most explored eucaryotic transformation technique at the biological and immunochemical levels. Genetic and biochemical information relating to the organization of the SV40 genome has been established or confirmed by the nucleotide sequence of the viral genome. Review by Tooze (1980), Molecular Biology of Tumor Viruses, 2nd ed., Part 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. The design of different SV40 vector molecules has relied on the accurate mapping of genetic signals and the use of restriction endnucleases for the isolation of defined fragments from the SV40 genome.
SV40 was developed initially as a eucaryotic-transducing vector using a lytic system. Mulligan et al., (1929), Nature (London), 277:108-114. Subsequently, transforming (nonlytic) vectors were constructed with isolated segments of the SV40 genome. Review of Elder et al., (1981), Annu. Rev. Genet., 15:295-340.
Hamer et al. were the first to suggest that SV40 might be used to clone genes for which no probe was available. They suggested double-stranded cDNA copies from a heterogeneous mRNA population could be "shotgunned" into an SV40 vector, and virus carrying the desired sequence could be identified by using a radioactive or fluorescent antibody.
Hamer et al. first reported the construction of an SV40 recombinant expression vector containing an expression marker in 1979. Hamer et al., (1979), Cell, 17:725-735. Their SV40 vector contained the viral DNA sequences from the BamHI endonuclease restriction site at 0.14 map units clockwise to the HaeII restriction site at 0.82 map units. In addition to the entire early gene A and the origin of viral DNA replication, the vector contained the viral promoter, leader, intervening sequence, 5' portion of the body and 3' terminal sequences for the viral late 195 mRNA. It did not contain 1660 base pair (bp) of late region sequences encoding the viral protein UPI, 2 and 3. Priers et al., (1978), Nature, 273:113-120 and Reddy et al., (1978), Science, 200:494-502.
Rabbit beta-globin gene coding sequences were ligated into the above vector as an expression marker. To determine whether rabbit beta-globin was being synthesized in monkey cells infected with their recombinant vector, Hamer et al., supra, used a radioimmunoassay capable of detecting as little as 1.0 nanogram of globin.
Although Hamer et al. were able to demonstrate positive evidence of beta-globin expression, they expressed several reservations as to the utility of the SV40/beta-globin recombinant system. First, since globin is only sparingly soluble, significant losses may have been sustained during the preparation of samples for measurement. Thus, the determination of the amount of globin in the infected cells may be in error by as much as 10-fold. Second, the assay cannot distinguish between authentic globin and other immunologically- related products, such as read-through protein or polypeptide fragments.
A factor that Hamer et al. did not address is the high degree of homology between all eucaryotic globins. This homology makes it difficult to distinguish vector-induced globin expression from globin endogenous to the host cell system.
B. Hepatitis B Virus Peptides and Anti-Polypeptide Antibodies
The hepatitis viruses are markedly different agents. They are grouped together strictly by virtue of the "target" organ they affect, the liver. Although a number of viruses affect the liver as part of systemic infections, the term "hepatitis viruses" is usually taken to mean Type A (HAV), Type B (HBV), and the non-A, non-B agents. Of the three types of viruses, HBV is by far the most explored at the biological, immunochemical, and clinical levels.
HBV is classified as a DNA virus and differs in many respects from all other families of DNA viruses. HBV is composed of an outer coat (more substantial than a membrane or envelope) consisting of protein, lipid, and carbohydrate, and bearing a unique antigen complex; i.e., the hepatitis B surface antigen (HBsAg). It also contains an inner-nucleocapsid with an antigenic specificity distinct from that of the surface antigen; i.e., the hepatitis B core antigen (HBcAg).
A soluble antigen, HBeAg, is also recognized in the art. This antigen is thought to consist of HBcAg polypeptides that are not assembled into HBV cores, and consequently have a unique antigenic specificity in the unassembled state.
In typical self-limiting acute HBV infections, the following serological markers appear sequentially in serum of an infected host: HBsAg, HBeAg, anti-HBC, anti-HBE, and anti-HBS. The appearance of anti-HBE signals the eventual loss of detectable HBsAg. This is true in all cases of self-limited acute HBV infection. Following the disappearance of HBS, there is a delay of from a few weeks to several months before the appearance of anti-HBS.
During chronic HBV infection, HBsAg and anti-HBC are present. The host's serum can show either the HBeAg or anti-HBE serological markers; i.e. the patient can either be HBeAg or anti-HBE positive.
Sensitive and specific radioimnunoassays and enzyme immune assays for several of the HBV markers are in wide use. These highly sensitive serologic tests have provided a basis for monitoring the appearance of virus and immune response markers during the course of HBV infection.
In the past few years, many studies have indicated that each serologic marker signifies specific viral events for host responses during HBV replication. The profile of serologic markers at various stages during the clinical course of disease can thus offer useful diagnostic and prognostic information.
The association between hepatitis B virus and human hepatocellular carcinoma (HCC), liver cancer, has been extensively studied, and seroepidemiological as well as histopathological findings strongly suggest that HBV is directly or indirectly involved in the etiology of liver cancer. A number of hepatoma cell lines have been derived from human HCC, and detection of HBV-specific DNA integrated into the genome of two such cell lines, PLC/PRF/5 and EPH3B, have been reported. However, in these cell lines, only the hepatitis B surface antigen has been expressed in tissue culture as a virus-specific gene product. Other markers of HBV such as hepatitis B core antigen, hepatitis BE antigen, and a DNA polymerase have not been detected.
Some DNA tumor viruses of animals can produce transformation through the action of viral genes that regulate the replication and integration of the viral genome, and transformed cells by such viruses can bear a T (tumor) or neo(new) antigen expressed by the transforming genes. Recent evidence for an antigen analogous to T antigen has been obtained in human hepatoma cells containing integrated HBV genes using the anti-complement immunofluorescent staining technique. This antigen has been designated HBV-associated nuclear antigen (HBNA). Wen et al., (1983) Infect. Immun., 39:1361-1367.
HBNA was detected in sera from several HBsAg-positive HCC patients, and expression of the antigen was demonstrated in both cell culture and tumor tissue. In addition, anti-HBNA antibodies were found in the sera of some HBsAg-positive patients with HCC. HBNA may represent the previously unrecognized expression of an HBV gene.
In 1979 Galibert et al., Nature, 281:646-650, reported the nucleotide sequence of the HBV genome, a circular DNA of about 3200 bases that is part double- and part single-stranded, a feature unique among viruses. The long or L strand (completely circular) of the genome was found to contain four reading frames large enough to account for viral proteins. These regions were termed S, C, P and X, and are shown schematically in FIG. 1.
Regions S and C have been found to contain the genes for HBsAg and HBcAg respectively. Region P is thought to code for a protein similar in size and amino acid residue content to a DNA polymerase. Region X was postulated by Wen et al., supra, to be one of the probable sources of the gene coding for HBNA.
The mere tentative assignment of functions for the genes in regions P and X demonstrates the gap that still exists in understanding the genetic organization and molecular biology of HBV. A reason for this gap has been the absence of an in vitro system for propagation of the virus.
Until recently, the only source of HBV was the serum of human patients. The failure of attempts to grow the virus in cell culture is the result of its very narrow host cell range.
One approach to the problem of producing HBV DNA and its gene products has been to use recombinant DNA technology. This technology enables the large scale production of the nucleic acid sequences that code for a particular viral protein.
Tiollais et al. (1981), Science, 213:406-411 reported transformation of E. coli with pBR322 containing the gene coding for HBsAg, and reported production of significant quantities of that isolated gene. Those workers also wanted to study the HBsAg gene's location within the HBV genome, and the factors that affected its expression into protein. To do this they constructed expression vectors containing the HBsAg gene for use in both bacterial and mammalian cells.
One of the expression vectors constructed by Tiollais et al., supra, achieved bio-synthesis of a protein in E. coli that contained HBsAg antigenic determinants. It was built by inserting a portion of the gene coding for HBsAg into the bacteriophage plac5-1UV5 so as to conserve the reading frame of natural HBV.
In order to study HBV gene expression in mammalian cells Tiollais et al., supra, constructed a series of HBsAg-expression plasmids by inserting transfection elements at various locations within the whole HBV genome. The transfection elements allowed the entire HBV genome to be integrated in several different orientations into the genome of mouse cells transformed with the vector. In creating the vectors in this way those workers attempted to use HBV's naturally occurring genetic control elements.
Only three of the six expression vectors reported by Tiollais et al., supra, produced expression of the desired HBsAg protein. Its production was detected by testing for its presence in tissue culture fluids using sheep anti-HBsAG antiserum. The other viral markers known at the time of the study (i.e., HBcAg, HBeAg and DNA polymerase) were not detected in the transformed cells.
At the time of the above Tiollais et al. study, the possible existence of Region X was known as was the possibility that it may code for a HBV protein. That is, it was known that the HBV genome contained a capacity to code for more proteins than had been previously associated with the virus.
This problem has been called genotype in search of phenotype. It is this problem, inter alia, that Wen et al., supra, were addressing when they postulated Region X as containing the gene coding for HBNA.
Prior to the above Tiollais et al. and Wen et al. studies, Sutcliffe et al., (1980) Nature, 287:801-805, demonstrated, inter alia, a general technique for solving this problem. They chemically synthesized a polypeptide from within the protein predicted by the nucleotide sequence of a viral gene whose protein product was unknown. Antibodies were raised to the polypeptide and were reacted against all the proteins made by cells infected with the virus. Using antibodies to portions of a predicted protein, Sutcliffe et al. detected a previously unknown and unrecognized viral protein product.
To date, the use of this or any other technique to unequivocally identify the protein product of HBV genome Region X has not been reported. This may be because while the general concept of preparing synthetic antigens and using them to induce antibodies of predetermined specificity has been described, there remains a large area of this technology that continues to defy predictability.
The reasons for this are several. First, protein amino acid residue sequences deduced from a genetic sequence are of a hypothetical nature unless the nucleotide sequence reading frame is firmly established because of the redundancy of the genetic code.
Second, a synthetic antigen does not necessarily induce antibodies that immunoreact with the intact protein in its native environment. Third, a host's natural antibodies to an immunogen rarely immunoreact with a polypeptide that corresponds to a short linear portion of the immunogen's amino acid sequence.